title: Flow cytometry-based binding assay for GPR40 (FFAR1; free fatty acid receptor 1).
abstract: GPR40 is a G protein-coupled receptor (GPCR) whose endogenous ligands have recently been identified as medium- and long-chain free fatty acids (FFAs), and it is thought to play an important role in insulin release. Despite recent research efforts, much still remains unclear in our understanding of its pharmacology, mainly because the receptor-ligand interaction has not been analyzed directly. To study the pharmacology of GPR40 in a more direct fashion, we developed a flow cytometry-based binding assay. FLAG-tagged GPR40 protein was expressed in Sf9 cells, solubilized, immobilized on immunomagnetic beads, and labeled with the fluorescent probe C1-BODIPY-C12. Flow cytometry analysis showed that C1-BODIPY-C12 specifically labels a single class of binding site in a saturable and reversible manner with an apparent dissociation constant of approximately 3 microM. The FFAs that activate GPR40 competed with C1-BODIPY-C12 binding; thus, medium- to long-chain FFAs could compete, whereas short-chain FFAs and methyl linoleate had no inhibitory effect. Furthermore, ligands that are known to activate GPR40 competed for binding in a concentration-dependent manner. All the ligands that inhibited the binding promoted phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 in human embryonic kidney (HEK) 293 cells that expressed GPR40 and [Ca(2+)](i) responses in mouse insulinoma (MIN6) cells that natively express GPR40; however, pioglitazone, a thiazolidinedione that failed to compete for the binding, did not activate ERK or [Ca(2+)](i) response. This study showed that a flow cytometry-based binding assay can successfully identify direct interactions between GPR40 and its ligands. This approach would be of value in studying the pharmacology of GPCRs.
authors.affiliation: Department of Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Hara
authors.forename: Akira
authors.initials: A
authors.lastname: Hirasawa
authors.forename: Qi
authors.initials: Q
authors.lastname: Sun
authors.forename: Taka-aki
authors.initials: TA
authors.lastname: Koshimizu
authors.forename: Chisato
authors.initials: C
authors.lastname: Itsubo
authors.forename: Keiko
authors.initials: K
authors.lastname: Sadakane
authors.forename: Takeo
authors.initials: T
authors.lastname: Awaji
authors.forename: Gozoh
authors.initials: G
authors.lastname: Tsujimoto
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D001667:Binding, Competitive; D001681:Biological Assay; D001896:Boron Compounds / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D007340:Insulinoma / Q000378:metabolism / Q000473:pathology; D007668:Kidney / Q000166:cytology; D008024:Ligands; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008954:Models, Biological; D010766:Phosphorylation; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; C473034:FFAR1 protein, human; D005456:Fluorescent Dyes; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D002118:Calcium